1. The Field of the Invention
The present invention relates to microfluidic devices having large numbers of small (between about 1 nl and about 100 nl) sample wells.
2. The Relevant Technology
The term “digital PCR” was first coined in 1999 by Burt Vogelstein (Vogelstein and Kinzler 1999). Limiting dilution was used to detect a minor fraction of altered DNA by diluting to the point of having only one DNA template in a given reaction volume. PCR was then run with molecular beacon probes in solution. Once amplified, the reaction volumes were fluorescently analyzed; wild type and mutated DNA were then quantified by relying on binary positive/negative calls.
Recently the concept of digital PCR has been miniaturized using microfluidics to limit the amount of DNA template rather than dilutions. One such application presented was multigene analysis of environmental bacteria using multiplex digital PCR (Ottesen et al. 2006). Quantitative population analysis of transcription factor expression has also been shown (Warren et al. 2006). Each of these microfluidic applications uses Fluidigm's Digital Array chip. Fluid is distributed into parallel dead-end channels using pneumatic pressure. A comb valve is then actuated, deflecting a membrane down to section off 1,200 isolated reaction chambers of 10 nl each. This chip is then thermocycled and analyzed using a microarray scanner.
Ottesen et al. and Warren et al.'s research was used for absolute quantification of sample. However, downsides do exist to their micro fluidic platform and detection scheme. One downside is the high cost for manufacturing the microchips and the expensive equipment required to valve their system to create individual wells. Another downside is the multicolor requirement needed with labeled probes.
DNA melting analysis as a complement to PCR was introduced in 1997 (Ririe et al. 1997). A dye is included in the PCR that fluoresces in the presence of double-stranded DNA, but not single-stranded DNA. After amplification, fluorescence is monitored as the double-stranded DNA product is slowly heated. When the double helix melts, fluorescence rapidly decreases. The negative first derivative of fluorescence with respect to temperature shows the melting temperature (Tm) as maxima. Recent advances in melting instrumentation (Herrmann et al. 2006) and saturating DNA dyes (Wittwer et al. 2003) allow detection of single nucleotide polymorphisms (SNPs). If the change is heterozygous, DNA heteroduplexes alter the shape of the melting curve (Reed et al. 2004). If the change is homozygous, the absolute temperature of the melting transition shifts (Liew et al, 2004). DNA melting analysis, when compared to existing PCR analytical techniques, is advantageous because it is less complicated, faster (less than 20 minutes for PCR and analysis), and prevents contamination of the sample and environment due to its “closed-tube” format (Zhou et al. 2004). The specific dye used determines the capabilities of the method; LCGreen® Plus detects homozygous and heterozygous sequences well and does not inhibit PCR (Wittwer et al. 2003).
Analysis of the melting transition is often sufficient for genotyping. However, unlabeled probes combined with asymmetric PCR provide even greater specificity over a smaller region, which may be necessary for variant discrimination (Zhou et al. 2005) and is commonly believed to be essential for clinical assays. A recent publication has demonstrated that DNA melting analysis is capable of being miniaturized, thus providing initial results for a microarray chip platform, reducing reagent costs (Sundberg et al. 2007).